Low Molecular Weight Deoxyribonucleic Acid Polymerase from Calf Thymus Chromatin

Abstract Replication of polydeoxynucleotide templates catalyzed by the low molecular weight DNA polymerase from calf thymus chromatin proceeds only in the presence of a complementary initiator, complementary deoxynucleoside triphosphate, and a divalent cation. The minimum chain length of an initiator for initiation of polydeoxyadenylate is 7. Kinetic studies show a single binding site for deoxynucleoside triphosphate and one binding site for the template initiator. The Km for monomers varies from 28 to 42 µm. The Km for the template is about 1000-fold lower than for the monomer and is not markedly affected by base composition. The low molecular weight DNA polymerase does not have an associated 3'- to 5'-exonuclease function, enhancing the possibility for replicative errors. Several error mechanisms have been detected. When a mismatched nucleotide is present at the 3' terminus of the initiator molecule reaction does occur, and the initiator molecule with the mismatched nucleotide at the 3' terminus is incorporated into the product chain. In the absence of the complementary deoxynucleoside triphosphate the enzyme carries out a addition reaction at the 3' termini of initiated templates. The limited addition reaction is stimulated by the presence of the deoxynucleoside triphosphate complementary to the template, requires the presence of an initiated template, increases with increasing concentration of deoxynucleoside triphosphate, and is higher in Mn2+-catalyzed reactions than in Mg2+-catalyzed reactions. Nearest neighbor analyses on the products of homopolymer replication show that the 3'-termini of both the template chain and the product chain are used for this limited addition. A conservative estimate of the fidelity of the low molecular weight DNA polymerase in the poly(dA) replication is obtained by the thymidine to cytosine or thymidine to guanine ratio in the product, where C or G is the mismatched monomer found as the 3' neighbor to T. The T/C ratio is about 120,000 and T/G is about 10,000 in the products of Mn2+-catalyzed poly(dA) replication. In the products of Mg2+-catalyzed poly(dA) replication, the T/C ratio is about 180,000 and the T/G ratio is about 100,000.